Enzymes have not only high catalytic activity but also stereospecificity for catalysis as well as substrate specificity and reaction specificity. The enzyme stereospecificity is almost absolutely strict with a few exceptions.
With the recent development of precision research, usage of optical isomers has become increasingly important in the field of medicines, agricultural chemicals, feeds, perfumes, etc. This is because optical isomers are often completely different in their biological activities. For example, in the case of thalidomide, its D(R)-isomer is not teratogenic, but the L(S)-isomer is strongly teratogenic. The practical use of its racemate as medicine caused the drug hazard scandal. Furthermore, when one of the antipodes shows effective biological activity and another not only shows no activity at all but also acts as a competitive inhibitor to the effective antipode, the raceme's biological activity is often reduced to one half of that of the effective antipode or lower. Therefore, the availability of optically pure antipode (by synthesis or resolution) has become an industrially important issue. For this purpose, such techniques have been widely used as to synthesize racemate and to optically resolve it effectively and much attention has been paid on the optical resolution with enzyme, which does not generate side products and a large amount of waste solution.
In general, L-amino acids are used widely and in large quantities as seasonings, food/feed additives, medical transfusion solution, etc. and, thus, their utilization value is extremely high. While L-amino acids are produced mainly by the direct fermentation method using microorganisms, there has been also known the optical resolution method to produce L-amino acids by (stereospecifically) hydrolyzing N-acyl-DL-amino acids with L-aminoacylase. This method has been used conventionally for the industrial production of L-amino acids that are hardly producible by the fermentation method. The L-aminoacylase used in this method is widely distributed in animals, plants, and microorganisms. The enzymes derived from various biological sources have been purified and elucidated of their enzymological properties. Also, since the N-terminal amino acid of many proteins are thought to be acetylated in vivo, L-aminoacylase is considered to function in regenerating amino acid from N-acetyl-amino acid produced by degradation of proteins. In addition, of these L-aminoacylases, the one acting on N-acyl-L-glutamic acid has been thought to participate in the biosynthesis of arginine (Fruh, H., Leisinger, T., J. Gen. 125, ppl (1981)).
On the other hand, since D-amino acids are non-proteinaceous amino acids, they have not been of practical interest for a long time. In nature, existence of D-amino acids is limited to small cyclic peptides, peptidoglycans of bacterial cell wall, and peptide antibiotics. However, it has been elucidated that D-amino acids are present in the bound form in the constitutive component of neuropeptides, tooth enamelum, eye lens, and cerebral proteins, and the elucidation of physiological significance of D-amino acids and the research on enzymatic methods for producing them have been actively pursued.
At present, resolution of DL-amino acids is carried out by physicochemical, chemical, and enzymatic methods. Among them, the enzymatic method is most convenient, and a method for continuously producing L-methionine from N-acetyl-DL-methionine using a bioreactor with immobilized L-aminoacylase has been industrialized. One of the methods for producing D-amino acids utilizes hydantoinase. This method comprises two enzymatic steps of D-carbamyl derivative formation by the action of D-specific hydantoinase on DL-5-substituted hydantoin as the starting material, which can be synthesized from aldehyde analog economically, and the subsequent action of D-amino acid carbamilase. Another known method for producing D-amino acids comprises hydrolyzing N-acetyl-DL-amino acids using D-aminoacylase Sugie, M. and Suzuki, H., Agric. Biol. Chem. 44, pp1089 (1980), Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee H., and Wang, Y., J. Enzyme Microb. Technol. 14, pp384 (1992)!. In spite of the importance of D-aminoacylase for the production of D-amino acids, its physiological significance and structural function have not been elucidated yet.
The first report of the presence of D-aminoacylase was made by Kameda et al. in 1952 in Pseudomonas sp. KT83 isolated from soil Kameda, Y., Toyoura, H., Kimura, Y., and Yasuda, Y., Nature 170, pp888 (1952)!. This enzyme hydrolyzed N-benzoyl derivatives of D-phenylalanine, D-tyrosine, and D-alanine. Since then, D-aminoacylases derived from the following sources have been reported: genus Pseudomonas Kubo, K., Ishikura, T. and Fukagawa, Y., J. Antibiot. 43, pp550 (1980), Kubo, K., Ishikura, T. and Fukagawa, Y., J. Antibiot. 43, pp556 (1980), Kameda, Y., Hase, T., Kanatomo, S., and Kita, Y., Chem. Pharm. Bull. 26, pp2698 (1978), Kubo, K., Ishikura, T., and Fukagawa, Y., J. Antibiot. 43, pp543 (1980)!, genus Streptomyces Sugie, M. and Suzuki, H., Agric. Biol. Chem. 42, pp107 (1978), Sugie, M. and Suzuki, H., Agric. Biol. Chem. 44, pp1089 (1980), genus Alcaligenes (Tsai, Y. C., Tseng, C. P., Hsiao, K. M., and Chen, L. Y., Appl. Environ. Microbiol. 54, pp984 (1988), Yang, Y. B., Hsiao, K. M., Li, H., Yano, Y., Tsugita, A., and Tsai, Y. C., Biosci. Biotech. Biochem. 56, pp1392 (1992), Yang, Y. B., Lin, C. S., Tseng, C. P., Wang, Y. J., and Tsai, Y. C., Appl. Environ. Microbiol. 57, pp2767 (1991), Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee, H., and Wang, Y. J., Microb. Technol. 14, pp384 (1992), Moriguchi, M. and Ideta, K., Appl. Environ. Microbiol. 54, pp2767 (1988), Sakai, K., Imamura, K., Sonoda, Y., Kido, H., and Moriguchi, M., FEBS, 289, pp44 (1991), Sakai, K., Obata, T., Ideta, K., and Moriguchi, M., J. Ferment. Bioeng. 71, pp79 (1991), Sakai, K., Oshima, K., and Moriguchi, M., Appl. Environ. Microbiol. 57, pp2540 (1991), Moriguchi, M., Sakai, K., Katsuno, Y., Maki, T., and Wakayama, M., Biosci. Biotech. Biochem. 57, pp1145 (1993), Wakayama, M., Ashika, T., Miyamoto, Y., Yoshikawa, T., Sonoda, Y., Sakai, K., and Moriguchi, M., J. Biochem. 118, pp204 (1995), Moriguchi, M., Sakai, K., Miyamoto, Y., and Wakayama, M., Biosci. Biotech. Biochem. 57, pp1149 (1993)!.
In addition, Tsai et al. and Moriguchi et al. characterized D-aminoacylase derived from bacteria belonging to genera Alcaligenes and Pseudomonas, and further elucidating the amino acid sequence of the enzyme protein and the base sequence of the gene thereof. Moriguchi et al. found that bacteria belonging to genera Alcaligenes and Pseudomonas produced three different kinds of D-aminoacylase in response to the change of inducers Wakayama, M., Katsuno, Y., Hayashi, S., Miyamaoto, Y., Sakai, K., and Moriguchi, M., Biosci. Biotech. Biochem. 59, pp2115 (1995)!.
Furthermore, Moriguchi et al. determined DNA sequences of genes coding for these D-aminoacylases derived from genus Alcaligenes, and compared them with those of L-aminoacylases derived from Bacillus stereothermophilus, humans and swine, reporting, as a result, a low homology in the gene structure between these D-aminoacylases and L-aminoacylases Wakayama, M., Katsuno, Y., Hayashi, S., Miyamoto, Y., Sakai, K., and Moriguchi, M., Biosci. Biotech. Biochem. 59, pp2115 (1995)!.
On the other hand, as to Actinomycetes, Sugie et al. reported the presence of D-aminoacylase in genus Streptomyces, but did not purify the enzyme to fully elucidate its properties Sugie, M. and Suzuki, H., Agric. Biol. Chem. 42, pp107 (1978), Sugie, M. and Suzuki, H., Agric. Biol. Chem. 44, pp1089 (1989)!.